Process for the production of new pepstatins having anti-pepsin activity

ABSTRACT

A process for producing new pepstatins having anti-pepsin activity by cultivating a pepstatin producing strain in a nutrient medium containing assimilable carbon sources and nitrogen sources, and extracting the new pepstatins from the cultivated broth or medium.

ilnited States Patent [191 Umezawa et al.

[451 Mar. 4, 1975 i 1 PROCESS FOR THE PRODUCTION OF NEW PEPSTATINSHAVING ANTI-PEPSIN ACTIVITY [75] Inventors: l-lamao Umezawa, Tokyo;Tetsuji Miyano, Nagoya; Kohtaro Funaishi,

Okazaki; Tomio Takeuchi, Tokyo; Takaaki Aoyagi, Fujisawa, all of Japan[73] Assignee: Zaidan l-lojin Biseibutsu Kagaku Kenkyu Kai, Tokyo, Japan[22] Filed: Aug. 9, 1973 [2!] Appl. No.: 387,139

Related U.S. Application Data [63] Continuation-impart of Ser. No.233,533 March 10.

1972. abandoned.

[30] Foreign Application Priority Data Mar. 26, 1971 Japan 46-17292 [52]U.S. Cl. 195/80 R, 260/1125 [51] Int. Cl C12d 13/02 [58] Field of Search195/80 R [56] References Cited UNITED STATES PATENTS 3,819,486 6/1974Murao et al. 195/80 R Primary E.\'aminerLionel M. Shapiro Attorney,Agent, or FirmBrady, OBoyle & Gates 2 Claims, 2 Drawing; FiguresCROSS-REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of our copending application Ser. No. 233,533,filed Mar. 10, 1972, now abandoned.

This invention relates to processes for the production of new pepstatingroup substances produced by pepstatin producing microorganisms.Umezawa, one of the said inventors, and his co-workers discoveredpepstatin as an agent effective against stomach ulcer. Its producingmicroorganisms are identified as Streptomyces testaceus (ATCC No. 21469)Hamada et Okami and Streptomyces argenteolus var. toyonakensis. (ATCCNo. 21468). Pepstatin is a pentapeptide containing N- acylatedisovaleric acid at N-terminal and free carboxylic acid at C-terminal.(J. Antibiotics, 23, 259-262, 1970, ibid., 23, 263265, 1970). Pepstatincan be obtained by cultivating a pepstatin-producing strain in anutrient medium containing peptone or other nitrogen source, andextracting from its cultivated broth to purify. Pepstatin can beobtained also as its metal-salts, amides or esters, as described in theJapanese Patent Application No. 4446166 by Umezawa et al. andcorresponding US. application Ser. No. 37,165, Filed May 4. 1970. Theinventors studied pepsin, inhibitors in the cultured broth ofthe abovepepstatin producing strains under various conditions and found more thantwo additional pepstatin-like substances which have antipepsin activityas pepstatin but differ from pepstatin on silica gel thin layerchromatography and in the amide forming fatty acid noiety of pepstatinon gas chromatography of the acid hydrolysates.

One of the new pepstatins; called pepstatin B, can be crystallized asfine needle form of its methyl ester from methanol solution. It melts atabout 254-255C and elementary analysis gives C 60.4%, N 9.74-, H 9.38%calculated to be C ,,H N 0,, (C 60.6%, H 940%, N 9.82%). Opticalrotation is [a],, 95.5 (C=O.5, acetic acid). It gives a blue color byRydon-Smith reaction as pepstatin and red color by hydroxylamine-ferricchloride reaction. The methyl ester of pepstatin B is moderately solublein dimethylformamide, dimethylsulfoxide and acetic acid, but hardlysoluble in water, chloroform, benzene, ethyl acetate and ether. It ismore soluble in methanol than pepstatin methyl ester (5-10 mg/cc).

The acid hydrolysate of pepstatin B methyl ester by HCl for 16 hours at105C was extracted with ether and applied to gas chromatography and twodimentional thin layer chromatography. From ether extract, n-caproicacid was found. From the aqueous layer, valine and alanine (2:l)'werefound in addition to 3-hydroxy-4-amino-6 methylheptanoic acid.Therefore, the structure of pepstatin B was calculated to be thencaproic acid amide analog of pepstatin having the structural formula:

i CH3 l f w i 1 T Wherein R is the n-caproyl group,

R is and R is COOH.

On silica gel thin layer chromatography with the solvent system(chloroform methanol acetic acid :4:1), the Rydon-Smith color reactionof pepstatin B methyl ester gave Rf value of0.39 and pepstatin methylester gave 0.35. The anti-pepsin activity of pepstatin B methyl estershowed the same as that of pepstatin methyl ester.

Thus we have discovered that pepstatin producing strains can producepepstatin-like: compounds wherein the fatty acid moiety is substitutedby n-caproic acid. The inventors also confirmed the presence of anotherpepstatin-like compound called pepstatin C containing isocaproic acid atthe amide forming moiety and its methyl ester showed the same RF valueas pepstatin B (0.39). Its structural formula is as above wherein r isthe isocaproyl groups I R, is R (i and R is COOH. A third pepstatin-likecompound whose methyl ester shows Rf 0.42 has also been found, Thisanti-pepsin principle was obtained as crystals of fine needle form andits acid hydrolysate gave the same amino acids and 5 fatty acids of O Con gas chromatographic analysis.

The new pepstatins of this invention can be defined as pepstatinscontaining C -C1 fatty acids, such as ncaproic acid or isocaproic aciddifferent from isovaleric acid.

According to this invention, new pepstatins can be obtained by thefollowing procedures: Pepstatin producing strain is inoculated in anutrient medium containing casein, skimmed milk and/or soybean meal asnitrogen source, and incubated for 3-10 days under ordinary aerobiccondition until anti-pepsin activity of the broth reaches maximum. Theactive principle can be extracted with n-butanol from cultured broth orits filtrate or with methanol from mycelical mass. The extract thusobtained is concentrated to syrup or to yellow precipitates. The Syrupis put into 5-20 volumes of water by drop-seise to form a precipitateand the precipitate is filtered. The precipitate is dried to a crudestate of the new pepstatins. The crude pepstatin is dissolved in loweralcohols and esterified by heating or standing for several hours at roomtemperature or higher temperature with a small amount of esterifyingcatalysts such as hydrochloric acid, sulfuric acid,paraa-eo-na-cwco-na-ca-eo-nfsima-ca-m -co-na-cr-1- toluene sulfonicacid, thionylchloride, phosphorouspentachloride, orphosphorousoxytrichloride etc. The

liquor, the methyl ester of original pepstatin was almost non-existentand the purified methyl ester of new pepstatins can be recrystallized asfine needle from this mother liquor.

The procedures in this invention includes the methods of obtainingalkaline salts of new pepstatins by alkaline hydrolysis of new pepstatinalcohol esters and the free form of the new pepstatins by neutralizationof the above alkaline hydrolysate. The alkaline hydrolysis can becarried out by the same procedure described in the patent application oforiginal pepstatin. (Japanese PatentApplication No. 46166/69). Thealkaline salts ofnew pepstatins, thus obtained as sodium sale ofpepstatin B is a white amorphous powder having a decomposition point of250255C and showing 50% inhibition of pepsin (ID at 0.055y and [D was0.05'y. In contrast to pepstatin, pepstatin B (free form) was amorphousform of white powder which was obtained by recrystalization frommethanol/H O (2:1). It decomposed at about 2l0220C. Specific rotation,[01] was 85C (C=l.0, methanol). lts infrared spectrum is shown in FIG 2.It is easily soluble in methanol (100 mg/ml), times as much as originalpepstatin. It is also soluble in aqueous butanol, ethanol,isopropylalcohol, dimethylformamide, dimethylsulfoxide, acetic acid orpyridine and slightly soluble in aqueous acetone, but hardly soluble indehydrated acetone, dehy drated butanol, chloroform, benzene, ether andwater.

The presentinvention will be explained more in detail by way of examplesbut is not limited by these examples.

EXAMPLE 1 A strain of Streptomyces testaceus (ATCC No. 21469) producingpepstatin was inoculated in a sterilized medium containing glucose 5.5%,soybean oil 2.0%, skimmed milk 4.5%, milk casein 5.0%, K HPO,

' formed was filtered. The precipitate was washed with 0.15 %,NaCl0.35%,M so.1 711 0 0.15% (pH 6.45) or 2900 1 volume and incubated at 2324Cfor1l2 hours with aeration and agitation. The broth contained antipepsinprinciple equivalent to 2260 y/cc of pepstatin. The pH of the broth wasadjusted to 2.5 with diluted sulfuric acid and methanol of 3600 l wasadded. After this liquor was stirred for 40 minutes at l520C, 4% offilter aid was added and filtered and washed. The methanol eluate of6300 1 thus obtained was adjusted to pH 10 by alkali and evaporatedmethanol in vacuo to yield aqueous residue of 1900 1. To this aqueousresidue, n-butanol of 800 l was added and pH of the mixture was adjustedto 7.0. After agitation for 30 minutes at 20C, butanol extract of 960 lwas separated. The pH of this butanol extract was adjusted to 3.0 andconcentrated in vacuo to yield concentrated syrup of 1. This syrup wasdropped into water of 800 l by drop weise with stirring for minutes andthe precipitates thus water and lyophilized to obtain yellow coloredcrude powder of new pepstatins of 7990 g. Anti-pepsin principleequivalent to pepstatin was contained in 64% of the powder.

The crude powder of 6080 g. obtained was dissolved in methanol of 33 1volume and added with active carbon of 1.25 kg to decolorize. To thisdecolorized methanol solution of 38 l, concentrated sulfuric acid of 125cc was added and stirred for 2.5 hours at 60C. The reaction mixture wasneutralized by addition of 0.63 l triethylamine and stirred for 120minutes at 50-60C,

and obtained white crystalline powder. This powder was washed and driedin vacuo to yield 3230 g. of new pepstatin methyl esters havingantipepsin activity (ID50) at 00563 Its decomposition point was at250-25lC and [011 is 90 (C=l.0, acetic acid).

This powder contained more than of pepstatin B methyl ester (Rf 0.39 onthin layer chromatography).

EXAMPLE 2 New pepstatin methyl ester of 3100 g. obtained in Example 1was dissolved in 130 l of 0.2NNaOH% methanol at 60C to saponify for 2hours. The reaction mixture was concentrated in vacuo to evaporatemethanol and muddy concentrate was obtained. The concentrate .wasdissolved in 20 l of water saturated n-butanol and added with 15 l ofwater and adjusted pH to 3.0 by diluted fulfuric acid. After stirringfor 20 minutes, upper butanol layer of 18 l was dropped into water of270 l by drop-weise. The precipitate thus obtained after agitation for60 minutes was filtered and washed with water. The washed precipitatewas dried in vacuo to give white powder of 2500 g. This powder showeddecomposition point at 205-2l0C and [(11 =-84 (C=l.0, methanol). [D (50%inhi- EXAMPLE 3 g. of new pepstatin methyl esters obtained in Example lwere dissolved in 4 l of absolute methanol and decolorized with 20 g. ofactive carbon. The methanol solution was kept to stand over night andobtained 24 g. of white crystals at first. 4.2 l of the methanolicmother liquor was concentrated to 1.3 l and precipitation occured. Thisprecipitate was crystalized after 60 minutes with agitation at 60C.Secondary crystalization over night gave white crystals of 5.5 g. assecondary crop. This second crystals were dissolved in 1.5 l of hotmethanol and treated with active carbon. The methanolic solution waskept to stand over night and obtained 12.0 g. of white crystals as thirdcrop.

The above three crystalline substances showed following physico-chemicalproperties:

The first crystals:

Decomposition point: 25025 1C [04],, 90.5 (C=0.5, acetic acid) Rf 0.35substance: Rf 0.39 substance 1:1 1:2

(thin layer chromatograph, chloroform:

methanol acetic acid 94:4:1)

The second crystals:

Decomposition point: 253254C [a],, 91.5 (C=0.5 acetic acid) Mainly Rf0.35 substance and tract of Rf 0.42 substance (thin layer chromatograph,chloroform: methanol acetic acid 95:4:1)

The third crystals:

Decomposition point: 254255C [a] 95.5 (C=0.5, acetic acid) Mainly Rf0.39 substance (pepstatin B) and trace of Rf 0.42 substance (thin layerchromatography, chloroform:

methanol acetic acid 95:4:1)

lnfrared spectrum is shown in FIG. 1

Elementary analysis (found); C 60.14%, H 9.38%, N

Moderately soluble in dimethylsulfoxide, demethylformamide and aceticacid, hardly soluble in methanol (5-10 mg/cc) but more soluble thanpepstatin methyl ester.

EXAMPLE 4 Pepstatin producing strain (Streptomyces argenteolus var.toyonakensis (ATCC No. 21468) was inoculated in a sterilized medium of130 1 containing glucose 6.0%., glycerine 2.0%, skimmed milk 4.0%, milkcasein 4.5%, K HPO 0.1%, NaCl 0.3%, MgSO,. 7H O 0.1% (pH 6.8) andincubated for 137 hours under the same conditions in Example 1. Thecultured broth of 125 1 containing anti-pepsin principle equivalent to170 g. of pepstatin was added with n-butanol of 60 l and stirred for 40minutes at room temperature. Afterwards, butanol layer, water layer andsolid layer (mycelial mass) were separated by centrifugation. Thebutanol layer was washed with water and concentrated in vacuo to 1.5 lof syrup. pH of the syrup was adjusted to 3.0 and added 7.5 l ofn-hexane to yield yellowish brown precipitate with stirring. Afterstirring for 60 minutes, the precipitate was filtered, washed and driedin vacuo, and yielded 164 g. of new crude pepstatin, having antipepsinactivity of ID 0.0757.

150 g. of new pepstatin was dissolved in 1.5 1 of 90% methanol at 40-50Cand decolorized by 150 g. of active carbon. 1.6 l of the decolorizedmethanol solution was added with 1.0 l of water at room temperature andyielded gel-precipitate. The precipitate was filtered,

washed with 50% methanol and lyophilized to give 94 g. of slightlyyellowish powder. Its lD against pepsin was 0.062 y. This powder wasdissolved in 9.0% of methanol and repeated twice to treat with activecarbon as above, and 45 g. of white new pepstatin was obtained. lts IDwas 0.055 y. It showed decomposition point at 198-205C and [011 79(C=l.O,.methanol). On thin layer chromatograph, it showed Rf 0.35(pepstatin), Rf 0.39 (pepstatin B and pepstatin C) and Rf 0.42.

EXAMPLE 5 Pepstatin producing strain (Srrepromyces testaceus ATCC No.21469) was inoculated in 130 l of a sterilized medium containing glucose6.0%, glycerine 2.0%, skimmed milk 1.5%, casein 1.5%, soybean meal 5.7%,K HPO 0.1%, NaCl 0.3% and MgSO 7H O 0.1% (pH 5.5) and incubated for 137hours under the same conditions in Example 1. To 110 l of the culturedbroth containing anti-pepsin principle equivalent to 132 g. of pepstatinwas filtered with addition of filter-aid 7 kg and adjusted to pH 2.0with diluted sulfuric acid, and

' lute methanol to give slurry which was then stirred for 40 minutes.Thereafter, the slurry was filtered and the cake was .washed twice with40 l of methanol to yield 140 l of aqueous methanol eluate. l of theabove filtered broth was added with 35 l of n-butanoland stirred for 15minutes, and separated 37 l of butanol layer from 73 l of water layer.140 l of above aqueous methanol eluate obtained from mycelial cake wasconcentrated in vacuo to 27 l of aqueous solution and was mixed with 37l of above butanol extract obtained from the broth filtrate. Thismixture was centrifuged to remove water layer and obtain 35 l of butanolextract. The butanol extract was adjusted to pH 3.0 and concentrated invacuo to 1.0 l of syrup. This syrup was dropped into 15 l of water bydrop weise and stirred for 60 minutes to yield precipitate. Theprecipitate was filtered, washed with water and dried at 6070C to give158 g. of yellowish powder of new pepstatin. Its 50% inhibition againstpepsin (lD was 0.072 7. The powder thus obtained was esterified inmethanol as described in Example 1 to give 62 g. of white crystallinepowder of new pepstatin methyl ester. lts 1D50 0.05 'y, decompositionpoint 250252C and [01],, 90.6 (C=0.5, acetic acid). On thin layerchromatography, it was found to contain mainly. new pepstatin methylester (Rf 0.39) and trace of pepstatin methyl ester.

30 g. of this new pepstatin methyl ester thus obtained was saponified in450 cc of 0.2N-NaOH-% methanol solution for 2 hours at 60C. The reactionmixture was adjusted to pH 8.5 with diluted HCl and concentrated to 150cc forming white precipitate. The precipitate was filtered, washed withmethanol and dried in vacuo to yield white powder 17.9 g.) of newpepstatin sodium salt having lD 0.05 y, decomposition point at 245 -249Cand [01 84% (C=l.0, methanol). Its 700 mg consumed 10.1 cc of 0.lN-HC1by titratron.

EXAMPLE 6 Pepstatin producing strain (Srreptomyces argenleolus var.royokensis ATCC No. 21468) was inoculated in a sterilized medium (130 1)containing glucose 6.0%, glycerine 2.0%, milk casein 4.2%. peptone 1.0%,K HPO 0.1%, NaCl 0.3% and MgSO,. 7H O 0.1 (pH 7.0) and incubated for132.5 hours under the same conditions in Example 1. The cultured brothof 130 1 containing anti-pepsin principle equivalent to l 1 1 g. ofpepstatin was treated in the same way described in Example 1 and yielded133 g. ofcrude new pepstatin, having lD against pepsin 0.076 y. Y

The yellow powder thus obtained was treated in the same way described inExample 1 and yielded 58.5 g. of new pepstatin methyl ester having ID0.051 y, decomposition point at 255256C and [01],, -95.5 (C=0.5, aceticacid). On thin layer chromatograph, it gave Rf0.35 and Rf0.39 1:1 butRf0.42 was not observed. When pepstatin preparation thus obtainedcontained pepstatin equal amount to new pepstatin (1:1).

EXAMPLE 7 Free acid form of new pepstatin (4.0 g.) obtained in Example 2was dissolved in cc of absolute methanol under heating and added 0.2 ccof cone. H SO This mixture was refluxed for 6 hours on a boiling waterbath and concentrated in vacuo to 50 cc. Theprecipitate thus formed wasfiltered, washed with ethanol and dried to give 1.6 g. of powder. Thepowder was recrystalized from absolute methanol solution (100 cc) andobtained 1.15 g. of white crystalline powder of new pepstatin ethylester (pepstatin B ethyl ester), having lD 0.054 -y, decomposition pointat 25 25 1 C and [01],, 90 (C=0.5, acetic acid).

EXAMPLE 8 EXAMPLE 9 3.0 g. of new pepstatin obtained in Example 2 wasdissolved in 100 cc of warm absolute n-butanol and added 0.2 cc of cone.H SO Esterification was made for 6 hours at 80C and concentrated invacuo to 2.0 cc containing precipitates. The precipitate was filtered,washed with n-butanol and dried to yield 800.0 mg of white powder. Thiswas recrystallized from 15.0 cc of n-butanol solution and obtained 340mg of white powder (new pepstatin B n-butyl ester) having ID 0.084decomposition point at 205207C and [01],, 88 (C=0.5, acetic acid).

EXAMPLE 10 Pepstanone A was found as a minor active component in thecrude pepstatin preparation ofEx. 1. On silica gel thin-layerchromatogram developed with chloroform, methanol, and acetic acid(92.5:6:1.5), pepstanone A gave an Rf value 0.45, while pepstatins gave0.15 detected by Rydon-Smith reagent. Pepstanone A was isolated bysilica gel column chromatography using the same solvent. Aftercrystallization with methanol, fine needles of pepstanone A wasobtained. It showed 78-86% ofthe pepsin-inhibitory activity of pepstatinA.

The new compound melted at 263-265C. The molecular formula wasestablished as C H O N (M-.W.

639), [Found C 61.99, H 9.81, N 11.03, Calcd.: C 61.94, H 9.61, N10.95]. The molecular weight was confirmed by mass spectrometry, [M*,m/e 639]. Pepstanone A gave positive reaction with Rydon-Smith and Brady(2,4-dinitrophenyl-hydrazine) reagents, but was negative to ninhydrin.Potentiometric titration, electrophoretic behavior, and color reactionssuggested that there is no free carboxyl or amino group. The UV [A 280nm (e 114)] and IR [v cm" absorptions suggested the presence of aketo-group, which accounts for the positive Brady reaction.

Pepstanone A was hydrolyzed with 6 N HCl at C for 15 hours. The aminoacide analysis indicated the presence of 1 mole of alanine, 2 moles ofvaline. and 1 mole of 4-amino-3-hydroxy-6-methylheptanoic acid. Thepresence of iso-valeric acid was confirmed by gas chromatography of thehydrolyzate after esterification of the acidic ether extract. However,there could not be found a Brady positive substance in the hydrolyzate.

The 2,4-dinitrophenylhydrazone of pepstanone A was obtained as yellowneedles, mp. 268-270C. It has still 37% of the activity of pepstatin.The results of the elemental analysis agreed with the formula C ,,H O Nfor the hydrazone. [Found C 56.86, H 8.01, N 14.63. Calcd. for C H O N(M.W. 819): C 57.13, H 7.99, N 15.37]. The UV absorption maximumappeared at 358 nm (6 19,800) in methanol.

Acid hydrolysis of the hydrazone yielded a yellow substance (v), whichshowed a positive ninhydrin reaction. Compound V was isolated by silicagel column chromatography using chloroform and methanol (9:1) and wascrystallized with mixed solvent of ethyl acetate and methanol, m.p.202-206C, k 350mm (6 18,300). The molecular formula was established as CH O N .2 HCl, [Found: C 41.26, H 5.30, N 18.34, Cl 18.37. Calcd. C40.85, H 5.54, N 18.32, Cl18.55]. The mass spectrum showed the parentpeak at m/e 309 (C H O N and the base peak at m/e 252 (MC H The NMRspectrum was taken in DMSO- (1 solution as internal TMS reference [0.98(8) (3H. doublet, J=5.5 Hz), 1.00 (3H, doublet, J=5.5 Hz), 1.6 1.8 (3H,multiplet), 2.21 (3H, singlet), 4.18 (1H. triplet, J=6.5Hz), 8.2 9.1(3H, aromatic H), 8 9.5 (broad NH)].

From the above results structure V was deduced. Then the C-terminus ofpepstanone A was suggested to be 3-amino-5-methylhexanone-2. The aminoacid sequence in pepstanone A was expected to be the same as that ofpepstatins. The mass spectra of pepstanone A and the O-trimethylsilylderivative (FIG. 1) unambiguously supported the sequence.

We claim:

1. A process for the production of pepstatin derivatives acting aspepsin inhibitors. which have the formula CH'-- CH CH (l. 3

I s CH OH I CH- CH 10 l v cit-cit on by adding water to said syrup, andthe pepstatin derivatives thus precipitated are purified by the aid ofmethyl esterification.

2. The process of claim 1 including the step of converting said methylester to an alkali metal salt or another alkyl ester thereof wherein thealkyl group contains from 2-4 carbon atoms.

1. A PROCESS FOR THE PRODUCTION OF PEPSTATIN DERIVATIVES ACTING ASPEPSIN INHIBITORS, WHICH HAVE THE FORMULA
 2. The process of claim 1including the step of converting said methyl ester to an alkali metalsalt or another alkyl ester thereof wherein the alkyl group containsfrom 2-4 carbon atoms.